qPCR Lentivirus Titration(Titer) Kit

Alias: LV900

Price contact : support@gentaur.com

Description
The Lentivirus Titration Kit provides a fast and simple method for titrating lentivirus. This kit employs a quick RNA extraction step and determines viral RNA using qRT-PCR. The whole assay could be completed in only 2 hours. The titer primers supplied in this kit are designed for all HIV-1 based vectors detection. With the help of the on-line titer calculator, titration of any lentiviral preparation could be easier than ever before.
 
Lentivirus Titration by qRT-PCR

The Lenti-X qRT-PCR Titration Kit provides a fast and simple method for measuring lentivirus titer. The kits use a quick RNA purification step before quantifying the number of lentiviral genome copies using qRT-PCR and SYBR® technologies.

Rapid Determination of Lentiviral Titer

Whereas traditional titration methods require up to 10 days to complete, these titration kits require only 4 hours and work with all HIV-1-based lentiviral vectors. Using qRT-PCR dramatically shortens the time interval between viral harvest and target cell infection, allowing you to perform both on the same day.

Achieve Consistent Lentiviral Transductions

A major advantage of lentiviral gene delivery systems is the ability to control the level of gene expression by adjusting the multiplicity of infection (MOI) to dictate the number of viral genomes that enter your target cells. These titration kits allow you to make a rapid correlation between the lentiviral copy number and lentiviral infectivity in your cell type, and ultimately allow you to control the expression level between experiments.

How It Works

The qRT-PCR procedure entails subjecting serial dilutions of your sample and a control RNA of known copy number (provided) to one-step qRT-PCR. The lentiviral copy number contained in your sample can then be determined by comparing its Ct value to the standard curve.

RNA purification and DNase I treatment steps are critical to the protocol so even viruses packaged using transient transfections, which always contain residual plasmid DNA, can be accurately quantified using this kit.

The kit includes sufficient material for 200 qRT-PCR reactions.

Advantages of  Lentiviral qPCR Titer Kit
  • no RNA purification -- saves time and eliminates inaccuracy
  • ready-to-use reagent mix -- reduces variability
  • qRT-PCR in one step -- more sensitive and accurate than other methods
  • no NTC amplification -- our titration kit is the only one on the market that completely eliminates NTC signal due to our unique primer design. Similar kits from other companies all give NTC amplification, which compromise accuracy for viral titration

User Manual:

Product Description

Lentivirus qPCR Titration Kit enables a simple, one step titration assay for lentiviral vectors. This kit employs a quick RNA extraction step that can be conveniently and directly coupled with a qRT-PCR, all of which can be completed within 2 hours. Designed to deliver high sensitivity and specificity,  Lentivirus qPCR Titration Kit ensures minimal non-specific background and better overall performance compared to similar kits.

Kit components:

Product Components

Volume

Part No.

Virus Lysis Buffer

800 μl

LV900-1

Reagent-mix*

1.0 ml

LV900-2

STD1 (Lentivirus Standard 1)

300 μl

LV900-3

STD2 (Lentivirus Standard 2)

300 μl

LV900-4

* Contains primers and reagents for Reverse Transcription.

Storage Conditions

Upon arrival, the Lentivirus qPCR Titration Kit should be stored at -20oC. Avoid repeated freeze-thaw cycles to retain maximal performance. The Lentivirus qPCR Titration Kit is stable for 1 year from the ship date when stored and handled properly.

Protocol Overview

Virus samples originating from both virus-producing cell line(s) or purified viral preparations can be used for determining the titer. The Virus Lysis Buffer provided in the kit helps extract viral RNA from the virus sample (extracted RNA is then referred to as “viral lysate”). The Reagent-mix and the 2X qPCR MasterMix (not part of the kit) are then employed with the viral lysate and the lentiviral standard(s) (STD1/STD2) to determine the threshold cycle (Ct) values via a qRT-PCR. The resulting Ct values are then used to calculate the lentiviral titer of the virus sample(s).

Protocol

1.              Sample Preparation: For purified viral samples: dilute the virus to the range of 106-108pfu/ml with 1X phosphate buffered saline prior to subjecting the virus to viral lysis. For viral samples directly from a virusproducing cell line: collect the culture medium and centrifuge it for 5 mins at 2000 g to remove cells/ debris. The resulting supernatant will be subjected to viral lysis.

2.              Viral Lysis: Add 2 μl of the diluted purified viral preparation or the supernatant (from step 1) to 18 μl of Virus Lysis Buffer and incubate at room temperature for 3 mins. This solution is now referred to as the viral lysate. The Ct value of the viral lysate will be used to determine the titer of the unknown viral sample. Please note in this step the viral sample has been diluted 1:10; therefore this dilution factor needs to be taken into consideration when calculating the titer.

3.              qRT-PCR Set-up: Set-up the reactions as shown below. Note that all reactions, except NTC, must be set in triplicates.

Component

Viral Lysate

Positive Control (STD1)

Positive Control (STD2)

Negative Control (NTC)

2X qPCR MasterMix

12.5 μl

12.5 μl

12.5 μl

12.5 μl

Viral Lysate (from step 2)

2.5 μl

-

-

-

STD1

-

2.5 μl

-

-

STD2

-

-

2.5 μl

-

Reagent-mix

10 μl

10 μl

10 μl

10 μl

Final volume per reaction

25 μl

25 μl

25 μl

25 μl

Total number of reactions

3

3

3

1

4.              qRT-PCR Program: Program the real-time qPCR instrument as shown below. (Note: this specific program was optimized for ABI® StepOne™ system).

Step

Temperature

Duration

Cycle(s)

Reverse Transcription

42oC

20 mins

1

Enzyme activation

95oC

10 mins

1

Denaturation

95oC

15 secs

40

Annealing/Extension

60oC

1 min